This invention relates to the isolation of monoclonal antibodies.
The immune system provides an important mechanism for the resistance to infectious agents. The antibodies that are produced in response to the foreign substance (antigen) by the immune system also provide a good source of reagents for treatment of disease and for diagnostic purposes. Antibodies thus produced by the immune system have the characteristic of being heterogeneous.
With the advent of hybridoma technology first developed by Kohler and Milstein, it is now possible to generate monoclonal antibodies which are essentially homogeneous compositions having uniform affinity for a binding site. The production of mouse hybridomas by these investigators is described in Nature 256, 495-497 (1975); and Eur. J. Immunol. 6, 511-519 (1976). According to this method, tissue-culture adapted mouse myeloma cells are fused to spleen cells from immunized mice to obtain the hybrid cells that produce large amounts of a single antibody molecule. The fusion generally is carried out in the presence of polyethylene glycol (PEG) as described by Galfe et al., Nature 266, 550-52 (1977), followed by selection in HAT medium (hypoxanthine, aminopterin and thymidine) as described by Littlefield, Science 145, 709-10 (1964). Immunization can be carried out with virtually any foreign antigen of interest. These can be, for example, hormones, proteins, cell-surface antigens, tumor markers, viruses, bacteria, parasites and the like. The use of hybridomas to provide a wide variety of clinically important antigens is illustrated by the extensive list and references cited, for example, by Sevier et al., Clin. Chem. 27 (11), 1797-1806 (1981).
Monoclonal antibodies also can be generated from human cells. See, for example, Rosen et al., Cell 11, 139-47 (1977); Olsson and Kaplan, Proc. Nat. Acad. Sci. USA 77, 5429-31 (1980); Croce et al., Nature (London) 288, 488-91 (1980); Eur. Pat. Appl. No. 44,722, Jan. 27, 1982; and Eur. Pat. Appl. No. 62,409, Oct. 13, 1982. Such antibodies should have better tolerance in human immunotherapy than the antibodies raised in mice.
Once the desired hybridoma is successfully cloned it may be desired to culture the cells on a large scale for the production of monoclonal antibodies in bulk quantities. Useful methods for such large scale cultivation of mammalian cells for producing monoclonal antibodies are described by Feder and Tolbert, Sci. Amer. 248 (1), 24-31 (1983).
Typically, the hybridoma cells are grown in conventional cell culture media such as, for example, RPMI-1640 or Dulbecco's modified Eagle's medium. The medium is usually supplemented with serum. It is customary to include up to 15% fetal calf serum, but bovine and horse serum also can be used.
Another method of monoclonal antibody production is described in U.S. Pat. No. 4,409,331, which involves growing the hybridoma cells encapsulated in semipermeable membranes. HAT selection can be avoided according to this method since the fusion process is viewed under a microscope and the unsuccessful fusions can be discarded based on this visual observation.
Yet another means of producing monoclonal antibodies involves culture of the hybridomas on hollow fiber membranes as disclosed by Calabresi et al., Proc. AACR and ASCO, p. 302 (1981).
A somewhat different approach to the preparation of hybridomas which can be used for the production of monoclonal antibodies is by the electrofusion or cell fusion system of Zimmermann and Vienken, J. of Membrane Biol. 67, 165-82 (1982), Springer-Verlag, New York; Zimmermann, Biochim. Biophys. Acta 694, 227-77 (1982); and Bischoff et al., FEBS Letters, 147 (1), 64-8 (1982), Elsevier Biomedical Publishing Co. According to this method, the cells to be hybridized are exposed to a low-level, non-homogeneous, high frequency electric field which orients the cells into "pearl necklace" chains. A direct current pulse is then applied which opens micropores in adjoining cell membranes. This allows intermixing of the cellular contents and results in cell fusion. The electrofusion method thus eliminates the chemical trauma of polyethylene glycol and the biological harm of viral fusion in creating hybridomas for monoclonal antibody production.
After production of the monoclonal antibodies by cell culture, it is usually desired to isolate or purify the antibodies from the medium. Various known methods of such isolation include, for example, ammonium sulfate precipitation, dialysis, affinity chromatography on protein A-Sepharose.RTM., ion exchange on DEAE columns such as, for example, DEAE-Sephacel.RTM., DEAE-Biogel.RTM. and DEAE Affi-Gel .RTM. Blue, or affinity chromatography on anti-Ig-agarose. These and other such conventional methods of purification of monoclonal antibodies are described by Goding, J. Immunol. Meth. 39, 285-308 (1980); and Bruck et al., J. Immunol. Meth. 53, 313-19 (1982).